It is well known that most of the bodily states in mammals, including most disease states, are affected by proteins. Classical therapeutic modes have generally focussed on interactions with such proteins in an effort to moderate their disease-causing or disease-potentiating functions. However, recently, attempts have been made to moderate the actual production of such proteins by interactions with molecules that direct their synthesis, such as intracellular RNA. By interfering with the production of proteins, maximum therapeutic effect and minimal side effects may be realized. It is the general object of such therapeutic approaches to interfere with or otherwise modulate gene expression leading to undesired protein formation.
One method for inhibiting specific gene expression is the use of oligonucleotides. Oligonucleotides are now accepted as therapeutic agents with great promise. Oligonucleotides are known to hybridize to single-stranded DNA or RNA molecules. Hybridization is the sequence-specific base pair hydrogen bonding of nucleobases of the oligonucleotide to the nucleobases of the target DNA or RNA molecule. Such nucleobase pairs are said to be complementary to one another.
In determining the extent of hybridization of an oligonucleotide to a complementary nucleic acid, the relative ability of an oligonucleotide to bind to the complementary nucleic acid may be compared by determining the melting temperature of a particular hybridization complex. The melting temperature (T.sub.m), a characteristic physical property of double helices, denotes the temperature (in degrees centigrade) at which 50% helical (hybridized) versus coil (unhybridized) forms are present. T.sub.m is measured by using the UV spectrum to determine the formation and breakdown (melting) of the hybridization complex. Base stacking, which occurs during hybridization, is accompanied by a reduction in UV absorption (hypochromicity). Consequently, a reduction in UV absorption indicates a higher T.sub.m. The higher the T.sub.m, the greater the strength of the bonds between the strands.
For use as therapeutics, oligonucleotides must be transported across cell membranes or be taken up by cells, and appropriately hybridize to target DNA or RNA. These critical functions depend on the initial stability of the oligonucleotides toward nuclease degradation. A serious deficiency of unmodified oligonucleotides which affects their hybridization potential with target DNA or RNA for therapeutic purposes is the enzymatic degradation of administered oligonucleotides by a variety of intracellular and extracellular ubiquitous nucleolytic enzymes referred to as nucleases. For oligonucleotides to be useful as therapeutics or diagnostics, the oligonucleotides should demonstrate enhanced binding affinity to complementary target nucleic acids, and preferably be reasonably stable to nucleases and resist degradation. For a non-cellular use such as a research reagent, oligonucleotides need not necessarily possess nuclease stability.
A number of chemical modifications have been introduced into oligonucleotides to increase their binding affinity to target DNA or RNA and resist nuclease degradation. Several publications describe the synthesis of 7-deaza-7-substituted-2'-deoxypurine nucleosides and their incorporation into oligonucleotides. Buhr et al., Nucleic Acids Research, 1996, 24, 2974; Seela and Thomas, Helv. Chim. Acta, 1995, 78, 94; Seela et al., Bioorg. Med. Chem. Lett., 1995, 5, 3059.
Other publications describe 7-deaza-7-substituted-2'-substituted purines, wherein the 7-substituent is bromo, chloro, cyano, alkyl, alkynyl, aryl or heteroaryl, and the 2'-substituent includes hydroxyl, alkoxyl or alkoxyalkoxyl.
International Publication Number WO 93/09127, published May 13, 1993, describes 7-deaza-7-substituted-2'-substituted purines wherein the 7-substituent is bromo, chloro, fluoro, cyano, alkyl or alkynyl, and the 2'-substituent is hydrogen, or a hydroxyl or alkoxy group.
U.S. Pat. No. 5,594,121, issued Jan. 14, 1997, discloses 7-deaza-7-substituted-2'-substituted purines wherein the 7-substituent is an aryl, heteroaryl or alkynylheteroaryl group, and the 2'-substituent is hydrogen, or a hydroxyl, fluoro or alkoxyl group.
European Patent Application Number 94810255.3, filed May 3, 1994, describes 7-deaza-7-substituted-2'-substituted purines wherein the 7-substituent is cyano or an alkynyl group, and the 2'-substituent includes a methoxyethoxy group.
Australian Patent Application AU-A-14398/88, published Oct. 13, 1988, describes 7-deaza-7-substituted-2'-substituted purines wherein the 7-substituent is halo, hydroxyl, mercapto, alkyl or alkylthio, and the 2'-substituent is hydrogen or hydroxyl.
While it has been recognized that nucleosides and oligonucleotides bearing base and sugar modifications would be useful, there remains a long-felt need for oligonucleotides with greater binding affinity, hence improved hybridization characteristics, and greater nuclease resistance. Such oligonucleotides are desired as therapeutics, diagnostics, and research reagents.